Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

中文翻译：

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菠菜（苋科）雄性偏向重复 DNA 序列的克隆和物理定位

菠菜（Spinacia oleracea Linnaeus，1753）是研究雌雄异株植物早期性染色体进化分子机制的理想材料。简并寡核苷酸引发的聚合酶链反应 (DOP-PCR) 技术通过从显微解剖的单染色体中富集特定的重复 DNA 序列来促进与反转录转座子相关的研究。我们使用显微解剖的菠菜 Y 染色体的 DOP-PCR 扩增产物进行基因组减法杂交以筛选性别偏向的 DNA 序列。筛选得到 55 个男性偏向 DNA 序列，长度为 30 576 bp，其中 32 个 DNA 序列（12 049 bp）包含重复 DNA 序列，包括 LTR/Copia、LTR/Gypsy、简单重复和 DNA/CMC-EnSpm . 在这些重复的 DNA 序列中，选择包含 Ty3-gypsy 反转录转座子片段的四个 DNA 序列（SP73、SP75、SP76 和 SP77）作为荧光探针，用于在雄性和雌性菠菜核型上进行杂交。对于每个同源物，SP73 和 SP75 的荧光原位杂交 (FISH) 信号主要在着丝粒及其周围区域捕获。使用SP76和SP77探针进行FISH，杂交信号主要出现在每个同源染色体对的假定着丝粒附近，长臂上存在零星信号。结果可作为研究菠菜性染色体进化中重复DNA序列功能的基础。对于每个同源物，SP73 和 SP75 的荧光原位杂交 (FISH) 信号主要在着丝粒及其周围区域捕获。使用SP76和SP77探针进行FISH，杂交信号主要出现在每个同源染色体对的假定着丝粒附近，长臂上存在零星信号。结果可作为研究菠菜性染色体进化中重复DNA序列功能的基础。对于每个同源物，SP73 和 SP75 的荧光原位杂交 (FISH) 信号主要在着丝粒及其周围区域捕获。使用SP76和SP77探针进行FISH，杂交信号主要出现在每个同源染色体对的假定着丝粒附近，长臂上存在零星信号。结果可作为研究菠菜性染色体进化中重复DNA序列功能的基础。